Saccharomyces cerevisiae cells from a stationary culture were permeabilized with 1% toluene, 4% ethanol and 0.075% Triton X-100. Not only sugars but also ATP, NAD(+), magnesium and inorganic phosphate must be simultaneously added to initiate the ethanol fermentation. The optimal pH for the fermentation was between 6.9 to 7.0. Sucrose was a better substrate than glucose. Ethanol fermentation was greatly stimulated by the addition of 1 mM arsenate. Under this condition, permeabilized cells continued to produce ethanol for more than one hour at the rate of 0.141 mmol ethanol/min/mg protein. Methanol inhibited the fermentation with intact cells but did not inhibit the one using permeabilized cells. In contrast, propanol inhibited fermentations both with intact and permeabilized cells.